Impact of pulsed-electric field and high-voltage electrical discharges on red wine microbial stabilization and quality characteristics.

AIMS: In this study, pulsed-electric fields (PEF) and high-voltage electrical discharges (HVED) are proposed as new techniques for the microbial stabilization of red wines before bottling. The efficiency of the treatment was then evaluated. METHODS AND RESULTS: PEF and HVED-treatments have been applied to wine for the inactivation of Oenococcus oeni CRBO 9304, O. oeni CRBO 0608, Pediococcus parvulus CRBO 2.6 and Brettanomyces bruxellensis CB28. Different treatment times (1, 2, 4, 6, 8 and 10 ms) were used at 20 kV cm(-1) for the PEF treatments and at 40 kV for the HVED treatments, which correspond to applied energies from 80 to 800 kJ l(-1) . The effects of the treatments on the microbial inactivation rate and on various characteristics of red wines (phenolic composition, chromatic characteristics and physico-chemical parameters) were measured. CONCLUSIONS: The application of PEF or HVED treatments on red wine allowed the inactivation of alteration yeasts (B. bruxellensis CB28) and bacteria (O. oeni CRBO 9304, O. oeni CRBO 0608 and P. parvulus CRBO 2.6). The electric discharges at 40 kV were less effective than the PEF even after 10 ms of treatments. Indeed, 4 ms of PEF treatment at 20 kV cm(-1) were sufficient to inactivate all micro-organisms present in the wines. Also, the use of PEF had no negative impact on the composition of wines compared to the HVED treatments. Contrary to PEF, the phenolics compounds were degraded after the HVED treatment and the physico-chemical composition of wine were modified with HVED. SIGNIFICANCE AND IMPACT OF THE STUDY: PEF technology seems to be an interesting alternative to stabilize microbiologically wines before bottling and without modifying their composition. This process offers many advantages for winemakers: no chemical inputs, low energy consumption (320 kJ l(-1) ), fast (treatment time of 4 ms) and athermal (ΔT ≈ 10°C).

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level.

AIMS: In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. METHODS AND RESULTS: Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. CONCLUSIONS: Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking.

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

Genetic characterization of strains of Saccharomycescerevisiae responsible for 'refermentation' in Botrytis-affected wines.

AIMS: Saccharomyces cerevisiae is responsible for alcoholic fermentation of wines. However, some strains can also spoil sweet Botrytis-affected wines. Three 'refermentation' strains were isolated during maturation. Characterization of those strains in regards to their fingerprint, rDNA sequence and resistance to SO2, which constituted the main source of stress in Botrytis-affected wines, was carried out. METHODS AND RESULTS: Refermentation strains could be clearly discriminated by interdelta fingerprinting. However, they exhibited close relationships by karyotyping. A part of RDN1 locus sequence was examined by using PCR-RFLP and PCR-DGGE. The resistance of refermentation strains to SO2 was performed by using real time quantitative PCR focusing on SSU1 gene. CONCLUSIONS: Results suggested that refermentation strains were heterozygote in 26S rDNA and their ITS1-5.8S rDNA-ITS2 region sequence revealed relationships with 'flor' strains. As described in the literature for flor strain, two out of three refermentation strains constitutively developed a higher level of SSU1 expression than the reference strains, improving their putative tolerance to SO2. Therefore, refermentation strains of S. cerevisiae had developed many strategies to survive during maturing sweet wines. SIGNIFICANCE AND IMPACT OF THE STUDY: Singularities in rDNA sequence and SSU1 overexpression revealed a natural adaptation. Moreover, genomic relationship between flor and refermentation strains suggested that stress sources could induced selection of survivor strains.